duct-double-resin-casting-followed-micro-computed-tomography-for-3d

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the ...

murine-neural-plate-targeting-utero-nano-injection-neptune-at

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously ...

Karolinska Institutet

Wnt5a is a morphogen that activates the Wnt/planar cell polarity (PCP) pathway and serves multiple functions during development. PCP signaling controls the orientation of cells within an epithelial plane as well as convergent extension (CE) movements. Wnt5a was previously reported to promote differentiation of A9-10 dopaminergic (DA) precursors in vitro. However, the signaling mechanism in DA cells and the function of Wnt5a during midbrain development in vivo remains unclear. We hereby report that Wnt5a activated the GTPase Rac1 in DA cells and that Rac1 inhibitors blocked the Wnt5a-induced DA neuron differentiation of ventral midbrain (VM) precursor cultures, linking Wnt5a-induced differentiation with a known effector of Wnt/PCP signaling. In vivo, Wnt5a was expressed throughout the VM at embryonic day (E)9.5, and was restricted to the VM floor and basal plate by E11.5-E13.5. Analysis of Wnt5a-/- mice revealed a transient increase in progenitor proliferation at E11.5, and a precociously induced NR4A2+ (Nurr1) precursor pool at E12.5. The excess NR4A2+ precursors remained undifferentiated until E14.5, when a transient 25% increase in DA neurons was detected. Wnt5a-/- mice also displayed a defect in (mid)brain morphogenesis, including an impairment in midbrain elongation and a rounded ventricular cavity. Interestingly, these alterations affected mostly cells in the DA lineage. The ventral Sonic hedgehog-expressing domain was broadened and flattened, a typical CE phenotype, and the domains occupied by Ngn2+ DA progenitors, NR4A2+ DA precursors and TH+ DA neurons were rostrocaudally reduced and laterally expanded. In summary, we hereby describe a Wnt5a regulation of Wnt/PCP signaling in the DA lineage and provide evidence for multiple functions of Wnt5a in the VM in vivo, including the regulation of VM morphogenesis, DA progenitor cell division, and differentiation of NR4A2+ DA precursors.

Wnt5a regulates ventral midbrain morphogenesis and the development of A9-A10 dopaminergic cells in vivo.

Lrp6 is generally described as a receptor required for signal transduction in the Wnt/beta-catenin pathway. Wnt5a, however, is a Wnt ligand that usually does not activate Wnt/beta-catenin but rather activates noncanonical Wnt signaling. We have previously shown that Lrp6 can inhibit noncanonical Wnt5a/Wnt11 signaling and that Lrp5/6 loss-of-function produces noncanonical gain-of function defects, which can be rescued by loss of Wnt5a. Here, we describe other phenotypes found in Wnt5a/Lrp6 compound mutant mice, including a worsening of individual Wnt5a or Lrp6 loss of function phenotypes. Lrp6 haploinsufficiency in a Wnt5a-/- background caused spina bifida and exacerbated posterior truncation. Wnt5a-/-Lrp6-/- embryos displayed presomitic mesoderm morphogenesis, somitogenesis, and neurogenesis defects, which are much more severe than in either of the single mutants. Interestingly these results reveal a further level of complexity in processes in which Wnt5a and LRP6 cooperate, or oppose each other, during mouse development.

Genetic interaction between Lrp6 and Wnt5a during mouse development.

Wnts are known to bind and activate multiple membrane receptors/coreceptors and to regulate dopaminergic (DA) neuron development and ventral midbrain (VM) morphogenesis. The low density lipoprotein receptor-related protein (Lrp6) is a Wnt co-receptor, yet it remains unclear whether Lrp6 is required for DA neuron development or VM morphogenesis. Lrp6 is expressed ubiquitously in the developing VM. In this study, we show that Lrp6(-/-) mice exhibit normal patterning, proliferation and cell death in the VM, but display a delay in the onset of DA precursor differentiation. A transient 50% reduction in tyrosine hydroxylase-positive DA neurons and in the expression of DA markers such as Nurr1 and Pitx3, as well as a defect in midbrain morphogenesis was detected in the mutant embryos at embryonic day 11.5. Our results, therefore, suggest a role for Lrp6 in the onset of DA neuron development in the VM as well as a role in midbrain morphogenesis.

Delayed dopaminergic neuron differentiation in Lrp6 mutant mice.

Endocytosis is increasingly understood to play crucial roles in most signaling pathways, from determining which signaling components are activated, to how the signal is subsequently transduced and/or terminated. Whether a receptor-ligand complex is internalized via a clathrin-dependent or clathrin-independent endocytic route, and the complexes' subsequent trafficking through specific endocytic compartments, to then be recycled or degraded, has profound effects on signaling output. This review discusses the roles of endocytosis in three markedly different signaling pathways: the Wnt, Notch, and Eph/Ephrin pathways. These offer fundamentally different signaling systems: (1) diffusible ligands inducing signaling in one cell, (2) membrane-tethered ligands inducing signaling in a contacting receptor cell, and (3) bi-directional receptor-ligand signaling in two contacting cells. In each of these systems, endocytosis controls signaling in fascinating ways, and comparison of their similarities and dissimilarities will help to expand our understanding of endocytic control of signal transduction across multiple signaling pathways.

The role of endocytosis in activating and regulating signal transduction.

Formation of the metazoan body plan requires a complex interplay of morphological changes and patterning, and central to these processes is the establishment of apical/basal cell polarity. In the developing nervous system, apical/basal cell polarity is essential for neural tube closure and maintenance of the neural stem cell population. In this report we explore how a signaling pathway important for nervous system development, Notch signaling, impacts on apical/basal cell polarity in neural differentiation. CSL(-/-) mouse embryos, which are devoid of canonical Notch signaling, demonstrated a neural tube phenotype consistent with cell polarity and convergent extension defects, including deficiencies in the restricted expression of apical polarity markers in the neuroepithelium. CSL(-/-) mouse embryonic stem (ES) cells, cultured at low density, behaved as wild-type in the establishment of neural progenitors and apical specification, though progression through rosette formation, an in vitro correlate of neurulation, required CSL for correct maintenance of rosette structure and regulation of neuronal differentiation. Similarly, acute pharmacological inhibition of Notch signaling led to the breakdown of neural rosettes and accelerated neuronal differentiation. In addition to functional Notch signaling, rosette integrity was found to require actin polymerization and Rho kinase (ROCK) activity. Disruption of rosettes through inhibition of actin polymerization or ROCK activity, however, had no effect on neuronal differentiation, indicating that rosette maintenance is not a prerequisite for normal neuronal differentiation. In conclusion, our data indicate that Notch signaling plays a role not only in differentiation, but also in organization and maintenance of polarity during development of the early nervous system.

Notch signaling maintains neural rosette polarity.

Notch signaling regulates a vast array of crucial developmental processes. It is therefore not surprising that mutations in genes encoding Notch receptors or ligands lead to a variety of congenital disorders in humans. For example, loss of function of Notch results in Adams-Oliver syndrome, Alagille syndrome, spondylocostal dysostosis and congenital heart disorders, while Notch gain of function results in Hajdu-Cheney syndrome, serpentine fibula polycystic kidney syndrome, infantile myofibromatosis and lateral meningocele syndrome. Furthermore, structure-abrogating mutations in  result in CADASIL. Here, we discuss these human congenital disorders in the context of known roles for Notch signaling during development. Drawing on recent analyses by the exome aggregation consortium (EXAC) and on recent studies of Notch signaling in model organisms, we further highlight additional Notch receptors or ligands that are likely to be involved in human genetic diseases.

The developmental biology of genetic Notch disorders.

Breaking symmetry in populations of uniform cells, to induce adoption of an alternative cell fate, is an essential developmental mechanism. Similarly, domain and boundary establishment are crucial steps to forming organs during development. Notch signaling is a pathway ideally suited to mediating precise patterning cues, as both receptors and ligands are membrane-bound and can thus act as a precise switch to toggle cell fates on or off. Fine-tuning of signaling by positive or negative feedback mechanisms dictate whether signaling results in lateral induction or lateral inhibition, respectively, allowing Notch to either induce entire regions of cell specification, or dictate binary fate choices. Furthermore, pathway activity is modulated by Fringe modification of receptors or ligands, co-expression of receptors with ligands, mode of ligand presentation, and cell surface area in contact. In this review, we describe how Notch signaling is fine-tuned to mediate lateral induction or lateral inhibition cues, and discuss examples from C.elegans, D. melanogaster and M. musculus. Identifying the cellular machinery dictating the choice between lateral induction and lateral inhibition highlights the versatility of the Notch signaling pathway in development.

Do as I say, Not(ch) as I do: Lateral control of cell fate.

Alagille syndrome is a genetic disorder characterized by cholestasis, ocular abnormalities, characteristic facial features, heart defects, and vertebral malformations. Most cases are associated with mutations in JAGGED1 (JAG1), which encodes a Notch ligand, although it is not clear how these contribute to disease development. We aimed to develop a mouse model of Alagille syndrome to elucidate these mechanisms.

Mouse Model of Alagille Syndrome and Mechanisms of Jagged1 Missense Mutations.

Editorial: Signaling pathways instruct the blueprint of life.

The liver fulfills central roles in metabolic control and detoxification and, as such, is continuously exposed to a plethora of insults. Importantly, the liver has a unique ability to regenerate and can completely recoup from most acute, non-iterative insults. However, multiple conditions, including viral hepatitis, non-alcoholic fatty liver disease (NAFLD), long-term alcohol abuse and chronic use of certain medications, can cause persistent injury in which the regenerative capacity eventually becomes dysfunctional, resulting in hepatic scaring and cirrhosis. Calcium is a versatile secondary messenger that regulates multiple hepatic functions, including lipid and carbohydrate metabolism, as well as bile secretion and choleresis. Accordingly, dysregulation of calcium signaling is a hallmark of both acute and chronic liver diseases. In addition, recent research implicates calcium transients as essential components of liver regeneration. In this review, we provide a comprehensive overview of the role of calcium signaling in liver health and disease and discuss the importance of calcium in the orchestration of the ensuing regenerative response. Furthermore, we highlight similarities and differences in spatiotemporal calcium regulation between liver insults of different etiologies. Finally, we discuss intracellular calcium control as an emerging therapeutic target for liver injury and summarize recent clinical findings of calcium modulation for the treatment of ischemic-reperfusion injury, cholestasis and NAFLD.

Calcium Signaling in Liver Injury and Regeneration.

Liver diseases constitute an important medical problem, and a number of these diseases, termed cholangiopathies, affect the biliary system of the liver. In this review, we describe the current understanding of the causes of cholangiopathies, which can be genetic, viral or environmental, and the few treatment options that are currently available beyond liver transplantation. We then discuss recent rapid progress in a number of areas relevant for decoding the disease mechanisms for cholangiopathies. This includes novel data from analysis of transgenic mouse models and organoid systems, and we outline how this information can be used for disease modeling and potential development of novel therapy concepts. We also describe recent advances in genomic and transcriptomic analyses and the importance of such studies for improving diagnosis and determining whether certain cholangiopathies should be viewed as distinct or overlapping disease entities.

Cholangiopathies - Towards a molecular understanding.

Corrigendum to "Cholangiopathies - Towards a molecular understanding" [EBioMedicine 35 (2018) 381-393].

WITHDRAWN: Editorial Introduction.

Cholangiopathies are an important group of liver diseases affecting the biliary system, and the purpose of this review is to describe how diseases in the biliary system can be studied in mouse models. A particular focus is placed on mouse models for Alagille syndrome, a cholangiopathy with a strong genetic link to dysfunctional Notch signaling. Recently, a number of different genetic mouse models based on various manipulations of the Notch signaling pathway have been generated to study Alagille syndrome, and we discuss the resulting phenotypes, and possible causes for the phenotypic heterogeneity among the various models. In the final section, we provide a more general discussion on how well mouse models can be expected to mimic human liver disease, as well as an outlook toward the need for new technologies that can help us to gain new insights from mouse models for liver disease.

Mouse Models for Diseases in the Cholangiocyte Lineage.

In the zone for liver proliferation.

Characterization of the progression of cellular states during human embryogenesis can provide insights into the origin of pediatric diseases. We examined the transcriptional states of neural crest- and mesoderm-derived lineages differentiating into adrenal glands, kidneys, endothelium and hematopoietic tissue between post-conception weeks 6 and 14 of human development. Our results reveal transitions connecting the intermediate mesoderm and progenitors of organ primordia, the hematopoietic system and endothelial subtypes. Unexpectedly, by using a combination of single-cell transcriptomics and lineage tracing, we found that intra-adrenal sympathoblasts at that stage are directly derived from nerve-associated Schwann cell precursors, similarly to local chromaffin cells, whereas the majority of extra-adrenal sympathoblasts arise from the migratory neural crest. In humans, this process persists during several weeks of development within the large intra-adrenal ganglia-like structures, which may also serve as reservoirs of originating cells in neuroblastoma.

Single-cell transcriptomics of human embryos identifies multiple sympathoblast lineages with potential implications for neuroblastoma origin.

Genetic loss and gain of function in mice have typically been studied by using knockout or knockin mice that take months to years to generate. To address this problem for the nervous system, we developed NEPTUNE (NEural Plate Targeting by  NanoinjEction) to rapidly and flexibly transduce the neural plate with virus prior to neurulation, and thus manipulate the future nervous system. Stable integration in >95% of cells in the brain enabled long-term overexpression, and conditional expression was achieved by using cell-type-specific MiniPromoters. Knockdown of  by using NEPTUNE recapitulated the phenotype of   embryos. We used NEPTUNE to investigate , mutations in which cause spinocerebellar ataxia type 5.  knockdown induced dose-dependent defects in the neural tube, embryonic turning, and abdominal wall closure, previously unreported functions for . NEPTUNE thus offers a rapid and cost-effective technique to test gene function in the nervous system and can reveal phenotypes incompatible with life.

Highly efficient manipulation of nervous system gene expression with NEPTUNE.

Reconstruction of heterogeneity through single cell transcriptional profiling has greatly advanced our understanding of the spatial liver transcriptome in recent years. However, global transcriptional differences across lobular units remain elusive in physical space. Here, we apply Spatial Transcriptomics to perform transcriptomic analysis across sectioned liver tissue. We confirm that the heterogeneity in this complex tissue is predominantly determined by lobular zonation. By introducing novel computational approaches, we enable transcriptional gradient measurements between tissue structures, including several lobules in a variety of orientations. Further, our data suggests the presence of previously transcriptionally uncharacterized structures within liver tissue, contributing to the overall spatial heterogeneity of the organ. This study demonstrates how comprehensive spatial transcriptomic technologies can be used to delineate extensive spatial gene expression patterns in the liver, indicating its future impact for studies of liver function, development and regeneration as well as its potential in pre-clinical and clinical pathology.

ArticleTotal : 2	Year
Publication title	2021
Publication title	2022

Article	Year
Wnt5a regulates ventral midbrain morphogenesis and the development of A9-A10 dopaminergic cells in vivo. PloS one\| PubMed ID: 18953410	2008
Genetic interaction between Lrp6 and Wnt5a during mouse development. Developmental dynamics : an official publication of the American Association of Anatomists\| PubMed ID: 19795512	2010
Delayed dopaminergic neuron differentiation in Lrp6 mutant mice. Developmental dynamics : an official publication of the American Association of Anatomists\| PubMed ID: 19795519	2010
The role of endocytosis in activating and regulating signal transduction. Cellular and molecular life sciences : CMLS\| PubMed ID: 22113372	2012
Notch signaling maintains neural rosette polarity. PloS one\| PubMed ID: 23675446	2013
The developmental biology of genetic Notch disorders. Development (Cambridge, England)\| PubMed ID: 28512196	2017
Do as I say, Not(ch) as I do: Lateral control of cell fate. Developmental biology\| PubMed ID: 28969930	2019
Mouse Model of Alagille Syndrome and Mechanisms of Jagged1 Missense Mutations. Gastroenterology\| PubMed ID: 29162437	2018
Editorial: Signaling pathways instruct the blueprint of life. Developmental biology\| PubMed ID: 29885286	2019
Calcium Signaling in Liver Injury and Regeneration. Frontiers in medicine\| PubMed ID: 30023358	2018
Cholangiopathies - Towards a molecular understanding. EBioMedicine\| PubMed ID: 30236451	2018
Corrigendum to "Cholangiopathies - Towards a molecular understanding" [EBioMedicine 35 (2018) 381-393]. EBioMedicine\| PubMed ID: 30314899	2018
WITHDRAWN: Editorial Introduction. Developmental biology\| PubMed ID: 30639158	2019
Mouse Models for Diseases in the Cholangiocyte Lineage. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 31016657	2019
In the zone for liver proliferation. Science (New York, N.Y.)\| PubMed ID: 33632834	2021
Single-cell transcriptomics of human embryos identifies multiple sympathoblast lineages with potential implications for neuroblastoma origin. Nature genetics\| PubMed ID: 33833454	2021
Highly efficient manipulation of nervous system gene expression with NEPTUNE. Cell reports methods\| PubMed ID: 34557863	2021
Spatial Transcriptomics to define transcriptional patterns of zonation and structural components in the mouse liver. Nature communications\| PubMed ID: 34857782	2021
Advances in genetic, epigenetic and environmental aspects of rare liver diseases. European journal of medical genetics\| PubMed ID: 34942406	2022
Outside influence: the extrahepatic duct as a source for bile duct regeneration. Hepatology (Baltimore, Md.)\| PubMed ID: 35006621	2022

Emma R. Andersson

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